The blood level of thioredoxin 1 as a supporting biomarker in the detection of breast cancer

Abstract

Background: There is a long-time unmet need for a means to detect breast cancer (BC) using blood. Although mammography is accepted as the gold standard for screening, a blood-based diagnostic can complement mammography and assist in the accurate detection of BC in the diagnostic process period of early diagnosis. We have previously reported the possible use of thioredoxin 1 (Trx1) in serum as a novel means to detect BC. In the present study, we validated the clinical utility of Trx1 to identify BC by testing sera from biopsy-confirmed cancer patients and women without cancer.

Methods: We have generated monoclonal antibodies against Trx1 and developed an ELISA kit that can quantitate Trx1 in sera. The level of Trx1 was determined in each serum from women without cancer (n = 114), as well as in serum from patients with BC (n = 106) and other types of cancers (n = 74), including cervical, lung, stomach, colorectal, and thyroid cancer. The sera from BC patients were collected and classified by the subjects' age and cancer stage. In addition to the Trx1 levels of BC patients, several pathological and molecular aspects of BC were analyzed. Test results were retrospectively compared to those from mammography. Each test was duplicated, and test results were analyzed by ROC analysis, one-way ANOVA tests, and unpaired t-tests.

Results: The mean level of Trx1 from women without cancer was 5.45 ± 4.16 (±SD) ng/ml, that of the other malignant cancer patient group was 2.70 ± 2.01 ng/ml, and that from the BC group was 21.96 ± 6.79 ng/ml. The difference among these values was large enough to distinguish BC sera from non-BC control sera with a sensitivity of 97.17% and specificity of 94.15% (AUC 0.990, p < 0.0001). Most Trx1 levels from BC patients' sera were higher than the cut-off value of 11.4 ng/ml regardless of age, stage, histological grade, type, and specific receptors' expression profile of BC. The level of Trx1 could rescue women from most cases of misread or incomplete mammography diagnoses.

Conclusion: These results indicated that the blood level of Trx1 could be an effective and accurate means to assist the detection of BC during the early diagnosis period.

Keywords: Biomarker; Blood; Breast Cancer; Diagnostic interval; Diagnostics; Early diagnosis; Mammography; Sensitivity; Specificity; Thioredoxin 1.

Fig. 1

Higher blood level of Trx1 in BC patients compared with women without cancer and other types of cancer patients. A Separation of BC patients from non-BC control, including women without cancer and patients from 5 different types of cancer by blood level of Trx1. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml). B ROC curve analysis to determine the ability of blood Trx1 level to differentiate BC from non-BC group. The sensitivity and specificity were 97.17 and 94.15%, respectively, with an AUC of 0.990 ± 0.005

Fig. 2

The effect of age on the level of blood Trx1. Breast cancer patients were divided into age groups of the 30s, 40s, 50s, and 60 and over, and their blood levels of Trx1 were compared. The enclosed box is a linear regression of Trx1 level dependency on age showing R² of 0.001. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml)

Fig. 3

The effect of breast cancer types on the blood level of Trx1. A The effect of pathological types of breast cancer. DCIS, ductal carcinoma in-situ; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; IMPC, invasive micropapillary carcinoma; MC, mucinous carcinoma; ITC, invasive tubular carcinoma. B The effect of molecular subtypes of breast cancer on the level of blood Trx1. TNBC, triple negative breast cancer. The enclosed boxes are linear regression of Trx1 level dependency on BC type and subtype. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml)

Fig. 4

The effect of hormone receptor expression profile on the blood level of Trx1. Trx1 levels from breast cancer patients were analyzed by the expression profiles of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Three-letter remarks on x-axis indicate positive (P) or negative (N) expression of ER, PR, and HER2 in order. For example, PPN indicates ER⁺, PR⁺ HER2⁻. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml)

Fig. 5

The effect of breast cancer stage and grade on the blood level of Trx1. Trx1 levels from breast cancer patients were analyzed by stage and grade of the cancer. A Effect of breast cancer stage. The number of patients at each stage were 2 at stage 0, 37 at stage 1, 50 at stage 2, 15 at stage 3, and 2 at stage 4. B Effect of breast cancer grade that was assessed by biopsy. The number of patients at each grade were 24 at grade 1, 51 at grade 2, and 31 at grade 3. The enclosed boxes are linear regression of Trx1 level dependency on stage and grade. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml)

Fig. 6

Complementary effect of blood Trx1 level on mammography for higher accuracy. BI-RADS category of subject’s mammogram and corresponding blood Trx1 level were analyzed together. A total of 103 biopsy-confirmed BC patients, and 42 women without cancer who whose mammograms were archived at CNUH were analyzed. The numbers on the X-axis indicate the categories of the BI-RADS scoring system. The dotted line indicates the cut-off value of Trx1 level (11.4 ng/ml). Filled circle (●), biopsy confirmed BC patients; empty circle (○), women without cancer. +